Method: Pulse labeling HDX NMR

Conditions: pH 6.3; 15.0 Celsius; Probes: 26

Related publication:
 PMID 7757007

Experiment details: "Pulse labeling experiments were performed in a QFM-5 rapid-mixing device. DHFR (3 mg/mL) was denatured and all exchangeable amide hydrogens were deuterated by incubation in 6 M D4-urea, 50 mM potassium phosphate in 99.9% D2O (pH 6.3) at 15 °C for 1 h. Refolding was initiated by rapid sixfold dilution in 50 mM potassium phosphate buffer, pH* 6.3, containing 0.2 mM K2EDTA and 1 mM P-mercaptoethanol for variable time periods (t,) prior to the labeling pulse. The labeling pulse was initiated by a twofold dilution into either 200 mM tris-HCI for pulse pH* values below 9.5, or 200 mM glycine for pH* values at and above 9.5; both buffers contained 0.2 mM K2EDTA and 1 mM 6-mercaptoethanol."

Protection threshold: Strongly protected within 13 ms

Sequence: MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLDKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR
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Method: Pulse labeling HDX NMR

Conditions: pH 6.3; 15.0 Celsius; Probes: 26

Related publication:
 PMID 7757007

Experiment details: "Pulse labeling experiments were performed in a QFM-5 rapid-mixing device. DHFR (3 mg/mL) was denatured and all exchangeable amide hydrogens were deuterated by incubation in 6 M D4-urea, 50 mM potassium phosphate in 99.9% D2O (pH 6.3) at 15 °C for 1 h. Refolding was initiated by rapid sixfold dilution in 50 mM potassium phosphate buffer, pH* 6.3, containing 0.2 mM K2EDTA and 1 mM P-mercaptoethanol for variable time periods (t,) prior to the labeling pulse. The labeling pulse was initiated by a twofold dilution into either 200 mM tris-HCI for pulse pH* values below 9.5, or 200 mM glycine for pH* values at and above 9.5; both buffers contained 0.2 mM K2EDTA and 1 mM 6-mercaptoethanol."

Protection threshold: Weakly protected within 13 ms

Sequence: MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLDKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

Method: Pulse labeling HDX NMR

Conditions: pH 6.3; 15.0 Celsius; Probes: 26

Related publication:
 PMID 7757007

Experiment details: "Pulse labeling experiments were performed in a QFM-5 rapid-mixing device. DHFR (3 mg/mL) was denatured and all exchangeable amide hydrogens were deuterated by incubation in 6 M D4-urea, 50 mM potassium phosphate in 99.9% D2O (pH 6.3) at 15 °C for 1 h. Refolding was initiated by rapid sixfold dilution in 50 mM potassium phosphate buffer, pH* 6.3, containing 0.2 mM K2EDTA and 1 mM P-mercaptoethanol for variable time periods (t,) prior to the labeling pulse. The labeling pulse was initiated by a twofold dilution into either 200 mM tris-HCI for pulse pH* values below 9.5, or 200 mM glycine for pH* values at and above 9.5; both buffers contained 0.2 mM K2EDTA and 1 mM 6-mercaptoethanol."

Protection threshold: Not protected within 13 ms

Sequence: MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLDKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR
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Method: Native exchange NMR

Conditions: pH 6.86; 25.0 Celsius; Probes: all

Related publication:
 PMID 7757007

Experiment details: "Exchange-out experiment at pH* 6.86 and 25 °C. A lyophilized, fully protonated sample of DHFR containing 1.5 molar excess of folate and appropriate buffer components was dissolved in D2O and a 2QF-COSY spectrum was recorded immediately. Following the acquisition of the initial 2QF-COSY spectrum (- 12 h), a second spectrum was recorded. Cross peaks for amide hydrogens that did not decrease in intensity by more than 25% in the second spectrum relative to the first were chosen."

Protection threshold: amids persisting to exchange for 12 hours

Sequence: MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLDKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR
 CLICK TO DOWNLOAD SEQUENCE IN FASTA